What do u think about my protocol for EMSA with antibodies? - EMSA experts out there??? (Dec/02/2008 )

Im going to do an EMSA with antibodies for the first time tomorrow. A fellow PhD has tried it before and its never worked so I was wondering what you thought about the protocol that im going to use?

Supershift with antibodies

Premix on ice:

1µl poly-dldC
4 µl 5x binding buffer or 2 µl 10x buffer
1 µl antibody or IgG as negative control
2 µl BSA (1 µg/ µl )
2 µl 100mM DTT
Add 14 µl H2O

Add on ice: 2-5 µg nuclear extract in 5 µl 1x buffer

Incubate for 20min at RT
Add 1 µl labeled oligo
Incubate for 20min at RT
Load on 4% Polyacrilamidgel

The result of your supershift depends very much on the antibody concentration. So make sure, the antibody you are using is especially for EMSA or that you put in adequately enough of your antibody. Otherwise you will not get the supershift. I am speaking from painful experience with the wrong antibody